Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citationsis incorporated herein by reference as though set forth in full.
Angiosperm plastids carry a relatively small genome (ptDNA), which is 120 kb to 160 kb in size and encodes ˜130 genes (Raubeson and Jansen 2005). These genes include some, but not all genes required for plastid transcription and translation. In addition, plastid genes encode photosystem, ATPase and NDH subunits, a gene involved in protein degradation and another in lipid biosynthesis (Wakasugi, et al. 2001). Transformation of the plastid genome is employed to probe plastid gene function in knockout plants and to beneficially alter processes localized to plastids, for example photosynthesis (Sharwood, et al. 2008, Whitney and Andrews 2001, Whitney and Andrews 2003), lipid biosynthesis (Madoka, et al. 2002) and the biosynthesis of vitamins (Apel and Bock 2009). Transformation is also used to incorporate novel genes in the ptDNA for the production of industrial enzymes and pharmaceutical proteins. For reviews see (Bock 2007, Daniell, et al. 2005, Maliga 2004).
Plastid transformation is based on homologous recombination between the ptDNA and ptDNA fragments in the vectors flanking a marker gene. Because plant cells contain hundreds to thousands of ptDNA copies, selective amplification of the transformed ptDNA copy is important for the recovery of transplastomic clones. According to the commonly used protocol, the tobacco leaves are bombarded with DNA-coated gold particles, then the leaves are cut into small pieces and transferred to a shoot regeneration medium containing spectinomycin. The selective medium suppresses greening and shoot regeneration of wild type cells and the transplastomic clones are identified as green shoots. The shoots regenerating from the bombarded leaves are chimeric. Genetically stable plants with a uniform population of transformed ptDNA (homoplastomic plants) are obtained by regenerating new shoots from the chimeric leaves. Typically two cycles of such purifying regeneration are required to obtain homoplastomic plants. Marker genes available in plastids for selective enrichment confer resistance to spectinomycin and streptomycin (aadA) (Svab and Maliga 1993), kanamycin (neo or aph(3′)IIa) (Caner, et al. 1993, Huang, et al. 2002) (Lutz, et al. 2004), chloramphenicol (Li, et al. 2011) or the amino acid analogues 4-methylindole (4MI) and 7-methyl-DL-tryptophan (7MT) (ASA2) (Barone, et al. 2009). Because the plants that are expressing marker genes have no visual phenotype, homoplastomic state can be verified only by DNA gel blot analyses.
It is clear that selectable marker genes conferring a visibly detectable phenotype are highly desirable. It is an object of the invention to provide such marker genes.